The larvae of Scarabaeidae beetles (grubs) cause significant damage to a variety of crops and ornamental plantings in the United States. The Japanese beetle, a species of this insect family, is one of the most significant of these pests with respect to ornamental lawns and turf grass. Adult insects lay their eggs in turf, with the resulting grubs feeding on the roots and subsequently destroying the plant. Severe infestations of the grubs can destroy entire lawns due to the continuing increases in numbers of grubs from year to year. One thousand Japanese beetles can lay eggs for 50,000 grubs. In the case of Japanese beetles the damage is then greatly extended due to the fact that the adults feed on a wide variety of agricultural and ornamental plants including fruit trees, grape vines, and roses.
Milky disease is a natural disease of Scarabaeidae beetles, caused by the bacterium Bacillus popilliae. It's called Milky disease because of the white appearance of the grubs, created by a large number of bacterial spores in the hemolymph or insect blood. To characterize the mode of action of Bacillus popilliae on Scarabaeidae larvae, several genes encoding proteins toxic to the larvae of Scarabaeidae insects have been isolated from Bacillus popilliae strains (J. Bacteriol. 179, 4336-4341 (1997); U.S. patent application 20020182693, herein incorporated by reference). In this application, we describe a novel Bacillus popilliae gene, cryhime1 (SEQ ID NO:4), that encodes a protein active against Scarabaeidae larvae.
The relationship between Bacillus popilliae and milky disease has been exploited to produce an effective biopesticide made from a spore powder of Bacillus popilliae. When compared with other biopesticides made from other Bacilli such as Bacillus thuringiensis, however, Bacillus popilliae biopesticides are extremely costly. Unlike Bacillus thuringiensis, Bacillus popilliae sporulation has not been achieved in vitro, so biopesticides made from Bacillus popilliae spore preparations are produced commercially in vivo by injecting the larvae with bacterial cells. The larvae are incubated until they develop a milky appearance, and then crushed and dried to give a spore powder.
By using Bacillus thuringiensis in place of Bacillus popilliae, significant advantages are realized. In addition to reduced cost as described above, other advantages include the fact that Bacillus thuringiensis is better characterized than Bacillus popilliae (i.e., many different strains and tools for Bacillus thuringiensis are available to achieve maximal biopesticide production) and Bacillus thuringiensis does not harbor vancomycin-resistance genes as do some Bacillus popilliae strains (Antimicrob. Agents Chemother. 44(3), 705-709, 2000). Vancomycin-resistance genes could be transferred to bacterial pathogens of humans from the sustained presence of Bacillus popilliae spores in the environment. In fact, it has been suggested that clinical isolates of Enterococcus facealis acquired vancomycin resistance by the transfer of a gene cluster from Bacillus popilliae spores (Antimicrob. Agents Chemother. 44(3), 705-709, 2000).
For these reasons, it is desirable to produce a Bacillus thuringiensis biopesticide that is toxic to Scarabaeidae larvae.